Neocartilage and methods of use

ABSTRACT

Disclosed are neocartilage compositions characterized by having multiple layers of cells, said cells being surrounded by a substantially continuous insoluble glycosaminoglycan and collagen-enriched hyaline extracellular matrix, and which neocartilage phospholipids are advantageously enriched in anti-inflammatory n-9 fatty acids, particularly 20:3 n-9 eicosatrienoic or Mead acid. 
     Also disclosed are methods of growing neocartilage in substantially serum-free growth media and methods of producing a conditioned growth media containing compounds effective to enhance neocartilage formation. 
     The neocartilage compositions are useful as implants and as replacement tissue for damaged or defective cartilage and as a model system for studying articular cartilage disease and response to natural and synthetic compounds.

BACKGROUND OF THE INVENTION

This application claims priority from U.S. Provisional ApplicationSerial No. 60/043369, filed Apr. 4, 1997, which is incorporated hereinby reference.

This invention relates generally to novel neocartilage compositionsuseful as implants and as replacement tissue for damaged or defectivecartilage, as model systems for studying articular cartilage disease andarticular cartilage response to natural and synthetic compounds and forthe isolation of cartilage derived substances useful in thebiotechnology industry.

More specifically, the invention concerns neocartilage, particularlyhuman neocartilage, having multiple layers of cells surrounded by asubstantially continuous insoluble glycosaminoglycan andcollagen-enriched hyaline extracellular matrix, and whose membranephospholipids are enriched in the anti-inflammatory n-9 fatty acids,particularly 20:3 n-9 eicosatrienoic or Mead acid. Also provided aremethods of producing neocartilage in vitro by growing chondrocytes insubstantially serum-free growth media. The invention further relates tomethods for producing conditioned growth media comprising compoundseffective to enhance neocartilage formation, and for the isolation ofcartilage derived substances.

Unlike most tissues, adult articular cartilage does not self-repair.Normal articular cartilage is hyaline cartilage comprising a distinctivecombination of cartilage-specific collagens (types II, VI, IX, and XI)and aggregating proteoglycans (aggrecan) which give it the uniqueability to withstand compressive forces.

Chondrocytes are the cartilage-specific cells which give rise to normalarticular cartilage tissue growth in vivo. Adult chondrocytes, however,have generally lost their potential to reproduce and generate newcartilage in vivo, although they are responsible for maintaining tissuehomeostasis.

Attempts to grow human articular cartilage using traditional cellculture methods such as growing chondrocytes on tissue-culture plasticsurfaces using serum-containing growth media have proved unsuccessful.Although serum (the non-red blood cell portion of blood, clotted andspun down) is known to be a potent mitogen to chondrocytes, theirculture in serum-containing growth media has been reported to result indedifferentiation of the chondrocyte phenotype.

It is also well known in the art that growing chondrocytes in monolayerson plastic culture vessels for prolonged periods leads to loss of theirspherical shape and the acquisition of an elongated fibroblasticmorphology. Reginato, et al., Arthritis & Rheumatism 37: 1338-1359(1994). Biochemical changes associated with this morphological changeinclude loss of the articular cartilage phenotype, e.g., loss of roundedcell shape, an arrest of cartilage-specific collagen and proteoglycansynthesis, the initiation of collagen type I and III synthesis, and anincrease in small non-aggregating proteoglycan synthesis. Reginato, etal., supra.

Adult human chondrocytes grown directly on tissue-culture plastic ingrowth media containing serum, attach to the plastic substrate and failto deposit an insoluble matrix enriched in glycosaminoglycan.Glycosaminoglycan is the proteoglycan component essential to thephysiological function of articular cartilage and is the hallmark ofhyaline tissue. The extracellular matrix initially produced by methodsusing serum-containing growth media is not enriched in glycosaminoglycanand resorption of the matrix material occurs as the cell culture ages.

Attempts to overcome chondrocyte dedifferentiation in vitro haveincluded culturing chondrocytes at high densities and growing them insuspension culture or on substrata that prevent cellular spreading andattachment to the tissue-culture plastic. Reginato et al. described amethod of growing human fetal chondrocytes cultured on polyHEMA-coatedplastic dishes in a serum-supplemented DMEM growth media. Arthritis &Rheumatism 37: 1338-1359 (1994). This method was successful atmaintaining the cartilage-specific phenotype but produced only nodulesresembling articular cartilage, not a continuous layer of articularcartilage tissue.

Kuettner described in U.S. Pat. No. 4,356,262, a method of producingbovine cartilaginous tissue from which an anti-invasion factor may berecovered. This method provided culturing a monolayer of chondrocytes athigh densities in a suspension of serum-containing growth media in aroller bottle. This method produced nodules of tissue having anextracellular matrix, but not a continuous layer of articular cartilagetissue.

Another method that prevents chondrocyte attachment to tissue cultureplastic is described in Kandel, U.S. Pat. No. 5,326,357. Kandeldescribed methods of reconstituting bovine cartilage tissue in vitro byseeding chondrocytes on a porous tissue culture insert substrate whichhad been coated with type I collagen to facilitate chondrocyteattachment and growth in a serum-containing growth media. The tissueculture insert is used to separate the chondrocytes from the tissueculture plastic. This method produced a continuous cartilaginous tissuehaving zones of elongated and spherical chondrocytes which resemblenative bovine cartilage.

Without a readily available replacement tissue, recent methods ofarticular cartilage repair have focused on biological resurfacing ofcartilage defects with either a prosthetic device or with livechondrocytes. Methods of in vivo articular cartilage repair includetransplanting chondrocytes as injectable cells or as a composition ofcells embedded in a three-dimensional scaffold. These methods, like invitro neocartilage production, have been less than completelysuccessful. One such repair method is autogenous chondrocytetransplantation. Vacanti et al., WO 90/12603. In this method, normalchondrocytes obtained from the patient are surgically removed, culturedto increase cell number and then injected into the defective site andsecured in place with a periosteal flap. Brittberg, M., et al., N. Eng.J. of Med., 331: 889-895 (1994). This method requires two separatesurgical procedures to complete.

Allograft transplant methods, which require a single surgery, useimplants made of donor chondrocytes seeded and grown on a natural orsynthetic three dimensional scaffold (Vacanti, et al., U.S. Pat. No.5,041,138; Gendlyer, EP 0739631 A2). In these methods, the natural orsynthetic three-dimensional scaffold is provided to give the cellculture structure and to mimic the natural extracellular matrix whilethe cartilage tissue is produced in vivo.

It has recently been shown, however, that neither the autogenous nor theallogenic transplant method results in consistent growth of articularcartilage in vivo, but rather results in chondrocyte dedifferentiationand formation of fibrocartilage. Because of the reduced aggrecan contentof fibrocartilage, it cannot withstand the same biomechanical stressesas articular cartilage. Fibrocartilage degenerates with use, and itsformation following joint repair may promote joint dysfunction andpermanent disability.

In addition to the clinical need for readily available replacementtissue, healthy articular cartilage is needed for use in model systemsfor studying articular cartilage disease and to evaluate chondrocyteresponses to growth factors, cytokines and pharmaceutical compositions.

Osteoarthritis, the most common form of arthritic disease, affectsalmost 16 million people in the United States alone. Osteoarthritis ischaracterized by the appearance of focal lesions at the cartilagesurface. With advancing age and disease progression, these changes areaccompanied by a marked reduction in proteoglycan content, extensivedestruction of the collagen framework, a marked increase in tissuehydration, and subsequent joint dysfunction.

Osteoarthritis appears to develop within the articular cartilage ofweight-bearing joints, particularly joints of the knee, hip, hand, andfoot. Under normal physiological conditions, cartilage homeostasis ismaintained by the resident chondrocytes. This highly specialized cellfunctions to synthesize, assemble, and remodel all components ofcartilage extracellular matrix, including aggregating proteoglycan aswell as collagens type II, VI, IX, and XI. Despite intensive researchefforts to ascertain the biological basis of osteoarthritis, itsdevelopment and progression remain poorly understood.

Recent studies attempting to characterize collagenolytic activity inhuman osteoarthritis indicate a clear need for a reliable alternative toanimal models for elucidating early biological events of diseaseprogression. Most animal tissues do not express the complexity ofenzymes that have been implicated in human disease. Thus, animal modelsare inadequate for evaluating the efficacy of potential diseasemodifying agents in human osteoarthritis.

A shortage of normal articular cartilage for studying articularcartilage disease and articular cartilage response to natural andsynthetic compounds exists because the only source of healthy articularcartilage currently available is from deceased adult donors which mayshow degenerative changes.

BRIEF SUMMARY OF THE INVENTION

Among the several objects of the invention, therefore, may be noted:

the provision of novel neocartilage compositions and uses thereof;

the provision of methods of producing such novel neocartilagecompositions and methods of producing conditioned growth media for usetherein, and the provision of cartilage specific components useful intissue engineering.

The neocartilage of the invention is useful, for example, as replacementtissue for damaged or defective cartilage and as a model system forstudying articular cartilage disease and response to natural andsynthetic compounds. Illustratively, surgical implants, or allografts,of the neocartilage were created in vitro and surgically attached tonatural cartilage in animal models to repair surgically created defects.

Briefly, therefore, the present invention is directed to neocartilagecharacterized by one or more of the following attributes: containingmembrane phospholipids enriched in Mead acid, containing membranephospholipids depleted in linoleic or arachidonic acid, beingsubstantially free of endothelial, bone and/or synovial cells, having aS-GAG content of at least about 400 mg/mg of OH-proline, beingsubstantially free of type I, III and X collagen, containing a matrixsubstantially free of biglycan, being enriched in high molecular weightaggrecan, being produced in vitro using serum-free growth medium, beingessentially free of non-cartilage material, and being characterized byhaving multiple layers of cells surrounded by a substantially continuousinsoluble glycosaminoglycan and collagen-enriched hyaline extracellularmatrix.

The present invention is further directed to methods of growingneocartilage in substantially serum-free growth media. Initially,chondrocytes are isolated and adhered to a surface using means effectiveto produce a monolayer cell culture. The cell culture is then grown in asubstantially serum-free cell culture to produce the neocartilage of theinvention and the neocartilage so produced.

The invention is also directed to a biological material containingessentially purified cartilage-specific macromolecules.

Also provided is a conditioned growth medium adapted for use in growingcartilage cell cultures which contain heparin-binding growth factors, atleast one of which is a cartilage-derived morphogenetic protein (Changet al., J. Biol Chem 269: 28227-28234).

In a further aspect of the invention, a set of implant materials areprovided. These materials include a reparative amount of Mead acidenriched neocartilage or neocartilage produced as described above. Alsoincluded are means for implanting and adhering the neocartilage in atarget tissue locus.

In yet other aspects of the invention, methods of producing aconditioned growth media comprising compounds effective to enhanceneocartilage formation and such conditioned growth media are provided,and methods of producing cartilage derived compounds and suchcartilage-derived compounds, using serum-free growth media, are alsoprovided.

In an additional aspect of the invention, a method of screening apharmaceutical for its capacity to modulate arthritic disease isprovided. In this method, the neocartilage as described herein isco-cultured with the pharmaceutical in an amount and under conditionseffective for determining whether characteristic indications ofarthritic modulation are observed in the neocartilage.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 in two parts, FIGS. 1A and 1B, shows gross morphology of humanneocartilage produced in vitro. Fetal chondrocytes grown underserum-free conditions to day 120 produce hyaline tissue that is roughly1.5-2 mm thick. Wet weights of 300-400 mg were obtained from materialgrown in 12 well dishes. FIGS. 1A and 1B, respectively, representlateral and birds-eye views of human neocartilage at day 90 of culture.The structural characteristics of this hyaline cartilage mimics that ofnative articular cartilage.

FIG. 2 is a graphical representation which shows growth curves for fetalchondrocytes grown in the presence (solid line) and absence (dashedline) of serum. DNA content was measured over a 60-day time course usingHoechst 33528 fluorescent dye, following papain digestion. Herring spermDNA was used as standard. Chondrocytes grown under the specifiedserum-free conditions go through the same number of doublings as thosecells grown in the presence of serum.

FIG. 3, in two parts, FIGS. 3A and 3B, is a graphical representationwhich shows that serum deprivation promotes neocartilage formation invitro: Glycosaminoglycan and collagen deposition by fetal humanchondrocytes. Chondrocytes were isolated from the upper region of theproximal tibia and distal femur of human fetal knees (18-21 wksgestation) by sequential enzymatic digestion. Cell suspensions were thenfiltered, counted, and seeded at high density (24 well clusters) inmedia containing 10% serum to allow adherence. Upon reaching confluence(day 7), the cultures were subdivided into two groups such that half ofthe dishes remained serum-free (dashed line), while the remaining disheswere maintained in serum-containing media (solid line). Growth mediawere supplemented with 50 μg/ml ascorbate at every media change, usuallyevery 72 h. Sulfated glycosaminoglycan (S-GAG) (FIG. 3A), andhydroxyproline (OH-proline) (FIG. 3B), content of the newly synthesized,insoluble hyaline matrix were measured as described hereinbelow.Chondrocytes grown under serum-free conditions produced at least 10-foldgreater amounts of proteoglycan (sulfated glycosaminoglycan) and morethan 2-fold greater levels of collagen (hydroxyproline) as compared toparallel cultures that were maintained in 10% FBS.

FIG. 4, in two parts, FIGS. 4A and 4B shows the morphologic appearanceof human neocartilage (day 56) following serum repletion (10% FBS). Day28 neocartilage was treated with 10% FBS until termination on day 56(FIG. 4B), at which point neocartilages were fixed and stained withsafranin-O to visualize aggrecan. FIG. 4A, day 56 control neocartilage(serum-free). Notice the marked decrease in metachromatic staining whichoccurred in response to repletion with 10% FBS (FIG. 4B). Also noticethe flattened and elongated phenotype of chondrocytes present at thesurface of the neocartilage. These changes suggest that in the absenceof mechanical stimulation, serum promotes autolytic resorption ofhyaline cartilage matrix, the mechanism of which requires further study.

FIG. 5, in six parts, FIGS. 5A-5F shows the morphologic appearance ofnative and neocartilage matrix following formalin fixation and paraffinembedding. Tissue sections were cut and stained with either safranin-O(FIGS. 5A, 5C and 5E) or pentachrome (FIGS. 5B, 5D, and 5F) to visualizespecific extracellular matrix components.

Safranin-O stains red and identifies S-GAG, whereas pentachrome stainsyellow for collagen, green for proteoglycan, and black for elastic.Because the tissues are enriched in collagen and proteoglycan, an aquablue color is achieved upon pentachrome staining.

FIGS. 5A and 5B show longitudinal sections of fetal proximal tibia usedto create the neocartilage grafts in FIGS. 5C-5F.

FIGS. 5C and 5D represent neocartilage grown under serum-free conditionsand harvested at day 90.

FIGS. 5E and 5F demonstrate the inhibitory effect of serum onneocartilage formation (day 0-30). (10% FBS).

Serum supplementation caused the cell layers to ball up and slough awayfrom the tissue culture surface after day 30. Note that FIGS. 5C and 5Dshow only a small fraction of the full thickness of day 90 neocartilage.Chondrocytes are seen localized to individual lacunae, forming hyalinetissue that is virtually indistinguishable in appearance from the nativestarting material and which spans 15-20 cell layers deep.Magnification×200 for FIGS. 5A, 5B, 5E and 5F, and×400 for FIGS. 5C and5D.

FIG. 6, in six parts, FIGS. 6A-6F shows the ultrastructuralcharacterization of neocartilage matrix by transmission electronmicroscopy (TEM). Representative cultures from FIG. 5 were fixed withglutaraldehyde, post fixed with osmium-tetroxide, and stained en-blocwith tannic acid and uranyl acetate. Ultra-thin sections werecounter-stained routinely with uranyl acetate and lead citrate. Highpower magnification×61,900 (B,D,F) of the ECM of native (FIGS. 6A and6B) and neocartilage tissue grown in either the presence (FIGS. 6E and6F), or absence (FIGS. 6C and 6D) of 10% FBS.

Note that FIG. 6E represents the entire thickness of the culturematerial and that these chondrocytes display cell/cell contact. Type IIcollagen (20 nm diameter fibrils) comprised the dominant structuralprotein in each of these tissues, confirming that the engineered tissueis hyaline in nature. Low power magnification×3,365 (A,C,E). A lack ofmatrix proteoglycan probably contributes to stacking of the collagenfibrils observed in FIG. 6F.

FIG. 7 shows SDS-PAGE and Western analysis of matrix associated collagenobtained by limited pepsinization and neutral salt precipitation.

S1 and S2 are duplicate samples of neocartilage tissue (day 90), whileFC and Sk represent native fetal cartilage and skin, respectively.

Antibodies to collagen types-II, IX and I were obtained from OncogeneSciences.

The analysis shows the presence of type II collagen and the absence oftype I collagen. Only the positive control (fetal skin) recognizedmonoclonal antibody directed to collagen type I.

FIG. 8, in two parts, FIGS. 8A and 8B, shows cytokine-induced resorptionof fetal articular cartilage. It is a demonstration of the relativeappearance of treated and untreated neocartilage cultures showing thatneocartilage exposed to cytokine is markedly reduced in size and liftsaway from the plastic surface.

Tissue was propagated as described above (day 28) and stimulated withincreasing concentrations of the indicated cytokine or growth factor(FIG. 8B) for an additional 36 days.

Cytokines were added to fresh ascorbate containing media every 48 hours.

Spent media was collected and frozen at each feeding for analysis ofS-GAG, hydroxyproline content, and matrix metalloproteinase (MMP)synthesis.

Likewise, remnant neocartilage was frozen for chemical analysis of ECMcomponents.

Note that in each case, the IL-1 and TNF-treated samples retractedconsiderably upon removal from the plastic surface (FIG. 8A), implyingthat increased synthesis and activation of the MMPs reduced thestructural integrity of the tissue.

FIG. 9, in four parts, FIGS. 9A-9D, shows a comparison of theextracellular matrix of control (FIGS. 9A and 9B) and activated (FIGS.9C and 9D) neocartilage following stimulation with interleukin-1. It isseen that chronic IL-1 stimulation alters cartilage staining forproteoglycan and collagen.

Day 60 neocartilages were treated with 1 ng/ml rhIL-1β for 30 days.Fresh media and cytokine were added every 48 hours.

Neocartilages were harvested (day 90) and stained with safranin-O andpentachrome as before. Staining intensity is markedly reduced instimulated (FIGS. 9C, 9D) versus untreated controls (FIGS. 9A, 9B).

Chondrocyte activation with IL-1 caused a significant reduction in thethickness of the synthesized matrix. Altered pentachrome stainingfollowing IL-1 stimulation suggests that MMP-mediated cleavage ofcollagen directly affects the binding characteristics of pentachrome dyeto collagen. Magnification×100.

FIG. 10, in two parts, FIGS. 10A and 10B, represent SDS-substrate gel(zymogram) analysis of cytokine-mediated production of matrixmetalloproteinases (MMPs) in fetal articular chondrocytes (neocartilagedisks). Negative staining indicates the molecular weight (MW) in kDa ofthe active proteases.

Spent media were collected 72 h post stimulation with the indicatedcytokine (5 ng/ml) and concentrated 16-fold by dialysis/lyophilization.

Unreduced samples were separated on 10% SDS substrate gels containingeither gelatin (FIG. 10A) or casein (FIG. 10B) 0.1%.

MMPs were renatured following SDS removal by extensive washing in 2.5%Triton X-100 and lytic bands visualized via negative staining withCoomassie blue, following incubation in a calcium and zinc containingbuffer for 4 hr at 37° C.

Latent enzyme activity is shown in lanes 1-4, while the correspondingactive enzymes were prepared by preincubation with 1 mM 4-aminophenylmercuric acetate (APMA lanes 5-8).

FIG. 11, in two parts, FIG. 11A and FIG. 11B, is an immunoblotdemonstrating cytokine-induced production of collagenase-1 (FIG. 11A)and collagenase-3 (FIG. 11B) in culture media.

Cell layers were stimulated chronically with 5 ng/ml rhIL-β and thespent media collected every 72 h.

Twenty μl fractions were reduced and immunoblotted with monospecificpolyclonal antisera to collagenase-1 (FIG. 11A) and collagenase-3 (FIG.11B).

Positive controls (purified recombinant protein) for each blot are shownin lane 1 and ranged in size from 50-54 kDa.

Media control, Lane 2; unstimulated control, lanes 3-4; 3-daystimulation, lane 5; 6-day stimulation, lane 6.

FIG. 12, in four parts, FIGS. 12A, 12B, 12C, and 12D, shows a surgicalmodel for articular repair. Arthrotomy was performed by a medialparapatellar incision and lateral patella dislocation. A full-thicknesscartilage defect, measuring 3 mm from proximal to distal traversing 5 mmfrom the medial edge of the medial femoral condyle, was created using aNo. 15 scalpel. The defect was curetted down to, while not violating,the subchondral bony plate. The medial side of the neocartilage graftwas sutured to the periosteum of the medial femur, while the lateralaspect of the graft was sutured to host cartilage at the lateral edge ofthe defect using 7-0 Proline sutures. After applying tissuetransglutaminase (Sigma Chemical Co.) under the graft via syringe(Jurgensen et al., J Bone Joint Surg, 79-A: 185-193, 1997), thegraft-defect interface was stabilized for five minutes via fingerpressure. The patella was then reduced, and the arthrotomy closed inlayers with interrupted 5-0 Ethibond suture. The skin incision wasclosed with interrupted subcuticular 5-0 Proline sutures. Medial andanterior aspects of the operated knee are depicted in FIGS. 12A, 12C,and in FIGS. 12B, 12D, respectively.

FIG. 13, in two parts, FIGS. 13A and 13B show the gross appearance ofrabbit knees at harvest following surgical implantation of rabbitneocartilage. Three by 5 mm (3×5) experimental defects were created inthe medial femoral condyles of 30 wk New Zealand White rabbits. Grossinspection six weeks post-operatively revealed that the neocartilageallograft was in place (FIG. 13B), and has taken on the appearance ofthe surrounding native cartilage. The unfilled defect of thecontralateral knee remained empty (FIG. 13A).

FIG. 14, in two parts, FIG. 14A and FIG. 14B, shows the results of twoseparate one-way mixed leukocyte assays used to measure alloreactivityvia lymphocyte proliferation. The experiment in FIG. 14B was set up toinvestigate alloreactivity of neocartilage, following enzymaticdissociation with collagenase and hyaluronidse. Legend: A=chondrocytesisolated from day 40 neocartilage (fetal donor); B=chondrocytes isolatedfrom day 30 culture (20 year male donor); C=chondrocytes isolated fromday 30 culture (27 year male); D=chondrocytes isolated from day 30culture (36 year female).

In FIG. 14A, 1×10⁵ irradiated peripheral blood leukocytes (PBL, AmericanRed Cross, St. Louis, Mo.) from two unrelated donors were mixed withnon-irradiated PBL from either the first (auto) or second donor (allo).Tritiated thymidine was added on day 6. Cultures were harvested and theamount of radiolabel incorporated into newly synthesized DNA counted.

In FIG. 14B, 1×10⁵ irradiated chondrocytes from four different donors of20-36 yrs age were incubated with 1×10⁵ non-irradiated PBL and theirproliferation measured on day 7. Chondrocytes failed to stimulateproliferation of allogeneic PBL obtained from donor 2. Positive (allo)and negative (auto) controls were run on the same plate and are includedfor comparison. Legend: PBL1=peripheral blood leukocytes from unrelateddonor 1; PBL2=peripheral blood leukocytes from donor 2.

FIG. 15 shows the co-stimulatory function of human neocartilagechondrocytes. 1×10⁵ T-cells, semi-purified from donor 2 (FIG. 14A) usingaffinity columns from R & D Systems, were incubated with irradiatedchondrocytes used in FIG. 14. Incubations were carried out at 37° C. forthree days. Again, chondrocytes failed to stimulate a proliferativeresponse above background.

FIG. 16 is a bar chart which shows that overlaying neocartilage withsecondary passage of chondrocytes increases allograft thickness andrigidity. Day 10 human neocartilage (12 well dishes) was overlayed with5×10⁵ chondrocytes obtained via secondary passage. These cultures wereharvested at day 28 and compared to control cultures that were initiallyseeded with 1×10⁶ cells/well.

FIG. 17, in two parts, FIGS. 17A and 17B, is a characterization ofneocartilage proteoglycans on 1.2% agarose gels. Human neocartilage (day35) was labeled for 72 hr with carrier-free sodium sulfate (10 μCi/ml).Matrix proteoglycans were then guanidine extracted, ethanolprecipitated, and extensively dialyzed prior to fractionation on 1.2%agarose gels. FIG. 17A, toluidine blue stained proteoglycan. FIG. 17B,autoradiograph showing localization of incorporated label in sixreplicates of neocartilage. Trace amounts of decorin were identified insix replicates of neocartilage, whereas biglycan, present in the 12- and43-year subjects, was not synthesized. Lanes: 1 (chondroitin-4-sulfatestandard); 2 (12-yr female); 3 (43-yr male); 4-9 (replicates of humanneocartilage). Note that native decorin can only be viewed in theneocartilage following metabolic labeling.

FIG. 18, in two parts, FIGS. 18A and 18B shows the effect of heparin onneocartilage formation. Day 10 neocartilage was treated with increasingamounts of heparin for 18 days. Fresh media, containing heparin andascorbate were added every 72-96 hrs. Media were collected and frozenfor future analysis. Cultures were harvested on day 28 and the S-GAGcontent (FIG. 18A) and OH-proline content (FIG. 18B) of the resultantneocartilage assayed as described hereinbelow.

FIG. 19 shows cartilage-derived morphogenetic proteins (CDMP's) in humanneocartilage matrix. Neocartilage formed by 90-day cultures ofchondrocytes from donors of various ages were extracted in a 1.2 Mguanidine-HCl buffer and passed over a heparin affinity column. Proteinswere eluted with 1 M NaCl and concentrated using a Centricon filter witha 10 K cutoff. The proteins were electrophoresed on a 12% SDS gel underreducing conditions, and transferred to Immobilon (Millipore) membranes.Immunoblots were probed with anti-CDMP antibody (N442) provided by theNIH. The secondary antibody was detected using chemiluminescence. Lanes:1 (fetal; 2 (1-day neonate); 3 (8-month infant); 4 (12-yr adolescent); 5(48-year adult). Immunoreactivity was detected in 56 Kd pre-forms, 34 Kddimers and 14-17 Kd mature proteins.

FIG. 20, in six parts, FIGS. 20A-20F, shows the fatty acid compositionof human neocartilage phospholipids versus time. Chondrocytes were grownin either the presence (open circles) or absence (filled circles) of 10%serum as described herein and the fatty acid composition of the membranephospholipids isolated via silicic acid chromatography determined viacapillary gas chromatography. The upper panels (FIGS. 20A, 20B and 20C),correspond to the n-6 polyunsaturated fatty acid precursors ofeicosanoid synthesis, while the lower panels (FIGS. 20D, 20E and 20F)designate the n-9 fatty acids which are abundant in rapidly growinghyaline cartilage. Notice that Mead acid (20:3 n-9 eicosatrienoic acid)accumulates under serum-free conditions to a level that was two-foldgreater than that identified in the native tissue at time zero.Additionally, serum supplementation caused a three-fold accumulation inarachidonate (20:4 n-6). The Mead-to-arachidonate ratio of serum vs.serum-free cultures mirrored that identified in adult and fetal tissue,respectively (Adkisson et al., 1991, supra). Samples were run induplicate.

FIG. 21 is a mass chormatogram showing the dominant polyunsaturatedfatty acid identified in neocartilage phospholipids (i.e. 20:3 n-9eicosatrienoic acid) at day 28 of culture. The fragmentation patternmatches that of authentic 20:3 n-9 eicosatrienoic or Mead Acid.

FIG. 22, in two parts, FIGS. 22A and 22B, shows the morphologicappearance of neocartilage/demineralized bone (Lambone) compositesfollowing pentachrome staining. Magnification: 22A, 100×; 22B, 200×.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, applicant has producedneocartilage tissue which has a morphological appearance largelyindistinguishable from healthy native articular cartilage. The cartilageproduced is strong, yet malleable. It is readily removable from culturevessels and can be grown with or without the aid of a three-dimensionalscaffold. Moreover, this novel cartilage has a membrane phospholipidfatty acid profile which is conducive to resistance to transplantrejection and inflammation. This newly developed biological materialalso serves as a ready resource for obtaining purified compositionsimportant for applications in biotechnology, includingcartilage-specific macromolecules such as high molecular weight aggrecanand collagen types II, VI, IX and XI.

The biological material hereby provided comprises neocartilage havingmultiple layers of cells surrounded by a substantially continuousinsoluble glyconsaminoglycan and collagen enriched hyaline extracellularmatrix. The collagen network of the neocartilage is randomly organized,and not separated into distinct zones of chondrocyte maturation. Thecollagen network of the neocartilage is randomly organized, and notseparated into distinct zones of chondrocyte maturation. Furthermore,the membrance phospholipids of the neocartilage are unexpectedly andadvantageously enriched in 20:3 n-9 eicosatriene (Mead) fatty acids anddepleted in linoleic and arachidonic fatty acids. The Mead acid contentis preferably at least about 0.4% and most preferably, between about0.4% and 10% of the total fatty acid content. The linoleic acid andarachidonic acid content is preferably less than about 0.5% of the totalfatty acid content of the membrane phospholipids. This neocartilage isfurther characterized by its high S-GAG and hydroxyproline content,enrichment in high molecular weight aggrecan, and the relative absenceof endothelial, bone and synovial cells, as well as being substantiallyfree of biglycan. Preferably, the neocartilage has a S-GAG content of atleast about 400 mg/mg of OH-proline, and more preferably, from about 800mg to about 2500 mg/mg of OH-proline. Preferably, the high molecularweight aggrecan contains at least about 80%, and more preferably, itcontains about 90%, of the total cartilage proteglycan content. Thechondrocytes of the neocartilage of the present invention aresubstantially spherical throughout the composition and maintain theirarticular cartilage phenotype. The neocartilage composition of thepresent invention is a substantially continuous layer of tissue at leasttwo cell layers thick. After 14 days of growth, the neocartilage hadgrown to between 10 and 15 cell layers thick. The neocartilage can begrown for at least 120 days to a thickness of at least 2 mm and to aweight of between 300 and 400 mg when 3.8 cm² dishes are used (15-20cell layers thick).

The neocartilage can be grown to substantially greater thicknesses whengrown under conditions that mimic the biomechanical forces to whicharticular cartilage is naturally subjected in vivo such as in acompression chamber. It has been found that after 30 days of growth, andas described herein, the glycosaminoglycan content of the neocartilageis approximately 600-1,500 mg S-GAG/mg OH-proline. This is 10 timesgreater than that of chondrocytes grown in the presence of 10% serum.

The neocartilage may comprise avian or mammalian chondrocytes,preferably human chondrocytes. Additionally, mammalian chondrocytes maybe derived from transgenic animals which have been geneticallyengineered to prevent immune-mediated xenograft rejection (Sandrin, M Set al., Nature Med 1: 1261-1267, 1995; Sandrin, M S et al.,Xenotransplantation 3: 134-140, 1996; and Osman, N et al., Proc Nat AcadSci 94: 14677-146821, 1997). Thus, the neocartilage may be mammalianneocartilage, including human and porcine, or avian neocartilage.

The use of organs/tissues from animal donors (xenotransplatation) is apotential solution to the chronic shortage of allogeneic organs. Porcinetissues are thought to be most suitable for human use due tosimilarities in size, anatomy and organ physiology between pigs andhumans. Recent insights into the mechanisms underlying vascularrejection, endothelial cell activation, and cellular responses toxenogeneic tissue have led to the development of novel strategiesdesigned to inhibit immune-mediated zenograft rejection (Dorling, A.,Expert Opinion on Therapeutic Patents, 7: 1307-1319, 1997).

The neocartilage of the present invention does not require the inclusionof biosynthetic polymer scaffolds in the composition. However, suchscaffolds can also be used, if desired. In one alternative embodiment,neocartilage can be grown on demineralized bone allograft forming acomposite which is particularly amenable to surgical implementation.

Because the neocartilage of the present invention can be produced freeof non-cartilage material, its use as an implant or as replacementtissue provides enhanced biocompatibility. For example scaffold-lessneocartilage readily integrates into the surrounding tissue whereascartilage constructs containing artificial polymer scaffolds are likelyto take longer to integrate because the cells must first break down theartificial scaffolds.

In another aspect of the invention, the neocartilage can be used as areplacement tissue for the repair of damaged or defective articularcartilage.

The replacement tissue can be mammalian or avian replacement tissue,most preferably human replacement tissue. Furthermore, mammalianreplacement tissue can be produced using chondrocytes from transgenicanimals which have been genetically engineered to preventimmune-mediated xenograft rejection.

The replacement tissue can be implanted using procedures well known inthe art, such as using traditional surgical means or by implantingorthoscopically.

Surgical implants comprising neocartilage can be surgically implantedand attached to natural cartilage in vivo by sutures or a combination ofsutures and biocompatible biological glues, such as tissuetrans-glutaminase (Jurgensen et al., J Bone J. Surg. 79A: 185-193.)

The neocartilage replacement tissue can also be attached to naturalcartilage in vivo by sutureless attachment such as chemical tissuewelding.

The neocartilage can be grown to various size specifications tofacilitate implantation.

Another embodiment of the invention provides using the neocartilage as amodel for studying articular cartilage disease and articular cartilageresponse to natural and synthetic compounds in vitro. Natural andsynthetic compounds of interest such as enzymes, cytokines, growthfactors, anti-invasion factors, dedifferentiation factors andpharmacologic agents are generally known in the art.

In particular, the neocartilage may be used in the testing ofpharmacologic agents useful in the treatment of diseases of the joint,for example, osteoarthritis and joint inflammation. Arthritis is markedby an increase in the synthesis and release of a variety ofcartilage-derived metalloproteinases and mediators of inflammation.Because these enzymes are directly responsible for tissue destruction inarthritis, the matrix metalloproteinases (MMPs) offer excellent drugtargets for the development of novel disease-modifying agents. In thisaspect, pharmaceuticals are screened for their capacity to modulatearthritic disease. The neocartilage is co-cultured with a candidatepharmaceutical and observed to determine whether characteristicsindicative of arthritic modulation are observed. The amounts andconditions employed are largely dependent on the particularpharmaceutical tested and employ methods well known in the art.

In yet another embodiment of the invention, applicant has discovered anovel method for producing neocartilage compositions in vitro fromchondrocytes. The method of this embodiment comprises:

isolating chondrocytes;

adhering the chondocyte cells to a surface in a manner effective toproduce a cell culture; this may be accomplished by growing thechondrocytes in a growth medium containing an amount of serum effectiveto allow adherence of the chondrocytes to an appropriate culture vessel;

replacing the growth media containing an amount of serum with asubstantially serum-free growth media to produce a substantiallyserum-free cell culture; and

growing the substantially serum-free cell culture to produceneocartilage and neocartilage-derived factors.

In a preferred method, chondrocytes isolated from immature donors suchas fetal, neonatal infant, or pre-adolescent chondrocytes are isolatedand grown in a substantially serum-free growth media to produceneocartilage.

The chondrocytes used in this method can be avian or mammalian,preferably human chondrocytes. Further, in contrast to other methods ofproducing neocartilage known in the art, such as seeding cells on threedimensional scaffold material or on material that prevents cellularspreading, further exogenous material is not required to produce threedimensional neocartilage. Unlike methods known in the art, the method ofthe present invention provides for seeding chondrocytes in directcontact with an appropriate tissue-culture vessel, most preferablyuncoated tissue-culture plastic. Although scaffold material isunnecessary, it can be used.

In a preferred embodiment of the invention, a cell culture is producedby isolating immature chondrocytes, e.g., fetal, neonatal, andpre-adolescent chondrocytes from donor articular cartilage.

Chondrocytes may be isolated by methods known in the art such as bysequential enzyme digestion techniques.

The isolated chondrocytes may then be seeded directly on a tissueculture vessel in a basal media comprising an effective amount of serumsuch as Dulbecco's modified Eagle's medium (DMEM) to allow adherence ofthe chondrocytes directly to the culture vessel and to promotemitogenesis.

The effective amount of serum added is between 2 and 15% fetal bovineserum, preferably 10%.

The culture medium may also comprise ascorbate, exogenous autocrinegrowth factors or conditioned growth media as described below.

The cell culture may be grown under suitable culture conditions known inthe art such as growing the cell culture at 37 degrees C. in ahumidified atmosphere with the addition of 2-10% CO₂, preferably 5%.

The growth media containing serum is replaced with growth mediumcontaining half as much serum, preferably 5% of the total growth mediumon between day 1 and day 10, preferably on day 7.

On between day 5 and day 14, preferably day 10, the serum containinggrowth medium is replaced with substantially serum-free growth media toproduce a substantially serum-free cell culture.

The preferred substantially serum-free growth media is HL-1, aserum-free media containing insulin-transferrin-selenium-complex as itsonly source of protein. HL-1 is a registered trademark of HycorBiomedical Inc., and also is available from BioWhittaker, Walkersville,Md. Other suitable serum-free growth media will be readily apparent tothose skilled in the art.

The substantially serum-free growth media is preferably partiallychanged periodically throughout the growth period. Following 10 moredays in the substantially serum-free medium, the neocartilage of theinvention is between 10 and 15 cell layers thick and can be removed fromthe cell culture with forceps as a rigid disk of neocartilage.

The neocartilage produced by the substantially serum-free cell culturecan be grown for at least 300 days. Even after 120 days in culture, thechondrocytes of the neocartilage do not dedifferentiate and fail tosynthesize collagen types I, III, and X. The method of the presentinvention can produce neocartilage of various sizes by using varioussized culture vessels.

In yet another aspect of the invention, a method for producing growthmedia is provided. The method comprises:

isolating fetal chondrocytes;

producing a cell culture by growth isolated chondrocytes in a growthmedia containing an amount of serum effective to allow adherence of thechondrocytes to an appropriate culture vessel;

replacing the growth media containing an amount of serum with asubstantially serum-free growth media after a beginning time interval toproduce substantially serum-free cell culture;

growing the substantially serum-free cell culture to produce conditionedgrowth media; and

extracting or concentrating the conditioned growth media from thesubstantially serum-free cell culture.

The conditioned growth media produced by the method of the presentinvention can be extracted by means such as heparin affinitychromatography. It may also be concentrated via dialysis andlyophilization. The conditioned growth media thus produced is reservedfor future use. The conditioned growth media obtained by this processcomprises compounds effective to enhance neocartilage formation such asautocrine factors, dedifferentintion factors, and anti-invasion factors.At least two of these autocrine factors have been identified byapplicant as cartilage-derived morphogenetic protein-1 and -2.

When the conditioned growth media is added to the substantially serumfree media to between about 5% and 30%, preferably at least about 20% ofthe total media, it increases the proliferation of neonatal and infantchondrocytes and subsequent deposition of the substantially continuousinsoluble glycosaminoglycan and collagen enriched hyaline extracellularmatrix.

In order to illustrate the invention in further detail, the followingspecific laboratory examples were carried out. Although specificexamples are thus illustrated herein, it will be appreciated that theinvention is not limited to these specific examples or the detailstherein.

The sources of various materials used in the specific laboratoryexamples are as follows:

MATERIALS

Dulbecco's modified Eagle's medium (DMEM) with added L-glutamine, sodiumpyruvate, and glucose (4.5 g/liter), fetal bovine serum (FBS) andantibiotic (100x) (penicillin G, sodium (10,000 units) and streptomycinsulfate (25 mg/ml of normal saline) were obtained from LifeTechnologies, Inc. (Grand Island, N.Y.).

Pronase-E (Type XIV, from Streptomyces griseus), hyaluronidase (typeIII), N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES), andMILLEX-GS syringe sterilization filters were obtained from SigmaChemical Company (St. Louis, Mo.).

Collagenase (CLS II) was purchased from Worthington Biochemicals(Freehold, N.J.). Tissue-culture dishes (12 and 24 well cluster) andbottle top sterilization filter units (type CA) were obtained fromCostar Corporation (Cambridge, Mass.).

Bovine serum albumin (fraction V, fatty acid-free) was from Calbiochem(San Diego, Calif.). HL-1 growth media, was obtained from HycorBiomedical (distributed currently by BioWhittaker).

EXAMPLE 1 Preparation and Culture of Immature and Adult HumanChondrocytes

Chondrocytes were isolated within 8-24 hours of death from the upperregion of the tibial plateau and femoral condyle of intact fetal bones(18-21 weeks gestation, Advanced Bioscience Resources, Inc., Alameda,Calif.). Hyaline cartilage from infant knees (≦8 months) was removedfrom the metaphysis of both the proximal tibia and distal femur usingrib cutters and transferred to sterile serum-free DMEM, containingantibiotics (2x) and 1% bovine serum albumin, for chondrocyte isolationas described below. Additional samples of more mature cartilage (12-58years) were processed similarly. All of these cartilage tissues wereprovided by Mid-America Transplant Association (St. Louis, Mo.). Alltissues were transported on wet ice prior to use.

Skin from fetal limbs was removed and stored at −20° C. for preparationof collagen standards (i.e., types I and III) via limited pepsinization.Skeletal muscle and other connective tissue were dissected under asepticconditions to expose the articulating surface of the tibia and femoralcondyle. The cruciate ligaments, menisci, and synovial capsule wereremoved.

Articular cartilage was separated from its underlying epiphysis (i.e.,more vascularized region), transferred to the cold synthetic cartilagelymph (SCL) described by Majeska and Wuthier, Biochim Biophys Acta,391:51-60 (1975), and washed extensively (4x) to remove contaminatingsynovial fluid. Any remaining connective tissue was removed from thecartilage by incubation with 1x trypsin-EDTA (Sigma Chemical Co.) for 30minutes at 37° C. The enzyme solution was replenished with freshserum-free DMEM and residual trypsin removed by four additional washesusing a vortex mixer.

At this stage of preparation, the cartilage was glistening white incolor and showed no evidence of fibrous tissue contamination. Cartilagewas then diced into 1 mm cubes, washed and transferred to 50 ml sterileconical tubes (approximately 2 g tissue/tube) containing 7 ml ofpronase-E solution (2 mg/ml Sigma Type XIV from S. griseus) in HL-1 for30 minutes digestion at 37° C. in an environmental incubation shaker setat 200 rpm (New Brunswick Scientific). The enzyme solution was removedand 4 ml of HL-1, containing 1 mg/ml BSA, antibiotics and ascorbate (50μg/ml) was added. To this solution, 2 ml of stock collagenase(Worthington CLS-II 1,000 units/ml in HL-1) and 1 ml of hyaluronidase(Sigma Type III, 5 mg/ml in HL-1) was added for overnight digestion at37° C. with mechanical agitation. The following morning cartilageremnants were diluted with 10 ml of fresh media and gently vortexed (3×1minutes) to release chondrocytes from the remaining extracellularmatrix.

Chondrocytes were then separated from tissue debris by gravityfiltration through a sterile Falcon cell strainer unit (70 μm) andsedimented at 600× g for 10 minutes in a clinical centrifuge. Cellviability was greater than 95%. Next, chondrocytes were diluted inDMEM+10% FBS for plating in either 12 or 24 well plastic culture dishesat a density of 1-2×10⁵ cells/cm² in 1 ml of growth media and grown at37° C. in a 95% air, 5% CO₂ atmosphere.

Chondrocytes were initially seeded in FBS to promote adherence and tostimulate cell division. Ascorbate (50 μg/ml, Sigma Chemical, St. Louis,Mo.) was added fresh at plating and at each feeding, generally every72-96 hours.

On day 7, neocartilage cultures were weaned of serum (5%) and remained100% serum-free (i.e., in HL-1) from day 10 onward. HL-1 is a chemicallydefined serum-free media containing insulin-transferrin-selenium-complexas its only source of protein. However, other serum-free media high inarginine and supplemented with insulin-transferrin-selenium may besubstituted for HL-1. Where indicated, conditioned media from fetalculture was added to 20% at all media changes to enhance neocartilageformation from infant chondrocytes.

Under the described serum-free conditions, immature chondrocytesdisplayed tremendous proliferative capacity, as well as the ability tosynthesize and deposit an insoluble hyaline cartilage matrix. A grossrepresentation of day 90 neocartilage tissue is shown in FIG. 1. Theneocartilage is rigid, yet malleable and was easily removed from culturevessels after day 20 using forceps. This material was strong enough tohold suture material after day 30 of culture and was amenable tosurgical implantation at day 40.

Neocartilage cultures were maintained under said conditions andharvested for biochemical and histological analyses between day 1-60 andagain at days 90 and 120. Parallel cultures were maintained in DMEM+10%fetal bovine serum for comparison.

EXAMPLE 2 Biochemical Assessment of Neocartilage Formation

Neocartilage formation was assessed by colorimetric analyses of sulfatedglycosaminoglycan (S-GAG) and hydroxyproline (OH-proline), generalmeasures of proteoglycan and collagen synthesis, respectively.Neocartilage disks were lyophilized and digested 18 hrs at 56° C. in 500μl of 0.1 M sodium acetate buffer (pH 5.6), containing 5 mM Na₄EDTA, 5mM L-cysteine, and 125 μg/ml papain. The digests were cooled to roomtemperature for determination of S-GAG, OH-proline, and DNA content.

Sulfated-GAG and the OH-proline content of papain digested material weredetermined at the indicated times via microplate colorimetric proceduresadapted from Farndale et al., Biochim Biophys Acta 883: 173-177 (1986),and Stegemann and Stalder, Clin Chim Acta 18: 267-273 (1967),respectively. Chondroitin-6-sulfate (Calbiochem) andcis-4-hydroxy-L-proline (Sigma) were used as standard.

DNA content was estimated by fluorometric analysis using a CytoFluormicroplate reader. Bisbenzamide (Hoechst 33258, Sigma Chemical Co., St.Louis, Mo.) was dissolved at 1 mg/ml in water and a working stockdiluted further to 0.1 μg/ml in 10 mM tris-HCl, pH 7.4, containing 0.1 MNaCl and 10 mM EDTA.

Ten, 20- and 50-fold dilutions of each sample were prepared and 10 μlaliquots assayed by mixing with 100 μl of dye solution. Fluorescence wasread (excitation wavelength=355 nm; emission wavelength=460 nm) and DNAcontent determined from a standard curve that was constructed usingherring sperm DNA (Gibco BRL).

Fetal chondrocytes displayed a rapid proliferative phase of cell growth(FIG. 2) and matrix deposition when grown under serum-free conditions,generating tissue that was 1.5-2.0 mm thick and weighing 300-400 mg (3.8cm² dishes) at day 120. In contrast, supplementation with 10% serumreduced total S-GAG and hydroxyproline content by 80-90% at day 30 (FIG.3). Furthermore, the structural integrity of these samples (+10% serum)was very poor. These samples could not be taken past day 30 of culturewithout shrinking and balling up. Day 28 neocartilage with 10% serum foran additional 28-day period causes >90% loss in matrix aggrecan (FIG.4). This effect was titratable, thereby suggesting that either specificcomponents of serum degrade cartilage matrix directly or, alternatively,that serum induces synthesis and/or activation of chondrocyte-derivedmatrix metalloproteinases.

The potential for neocartilage formation from pre-adolescent and adultarticular chondrocytes is depicted in Table I and appears to correlatewith skeletal development. Neocartilage formation was not recapitulatedin these experiments using articular cartilage obtained frompost-adolescent subjects.

TABLE I Effect of Donor Age on Neocartilage Potential Time in DonorCulture S-GAG Age (days) (μg) Fetal #1 30 3,583 ± 106   Fetal #2 609,126 ± 30   Fetal #3 120  7,750 ± 125   Infant (1 d) 25 6,853 ± 79  Infant (8 mo) 300  9,664 ± 313   12 y 30 1,102 ± 117   20 y 30 107 ± 15 27 y 30 46 ± 8  36 y 30 53 ± 13 43 y 30 31 ± 2  High density cultureswere established in 12 well clusters as described, and neocartilageformation was allowed to proceed until time of harvest. Triplicatesamples were assayed for total S-GAG using dimethyl methylene blue.

Characterization of the morphological appearance and biochemicalcomposition of the tissue revealed an ultrastructural organization thatwas hyaline in nature and nearly indistinguishable from native articularcartilage. Chondrocytes were constrained to individual lacunae and wereencased in an extensive extracellular matrix which stainedmetachromatically for aggregating proteoglycan (safranin-O) and collagen(pentachrome) (FIG. 5). Moreover, the pentachrome technique failed toidentify elastic fibers previously shown to be present in cultures ofbovine articular chondrocytes maintained in the presence of serum [Leeet al., Dev. Biol. 163: 241-252 (1994)].

Fibrocartilage contamination, a problem typically encountered whenarticular chondrocytes are cultivated using traditional cell culturemethods (i.e., media containing 10% serum), could not be identified inpepsinized extracts of neocartilage material by Western analysis(chemiluminescence detection) or by transmission electron microscopy(TEM) following tissue fixation. The dominant collagen identified by TEMconsisted of 20 nm fibrils, while beaded filaments, indicative of typeVI collagen, were localized to the lacunae (FIG. 6). Type I collagenfibers typically display a fibril diameter of 100 nm. Western analysisconfirmed the presence of type II collagen as the dominant (90%)isotype, and further identified minor cartilage-specific collagens suchas collagen types IX and XI (FIG. 7).

Cross reactivity with antibodies to collagen types III and X were alsonegative in neocartilage, but were reactive in collagen preparationsobtained from adult articular cartilage. Thus, the composition of thecollagen contained within the neocartilage tissue produced by the methodof the invention is indistinguishable in appearance from the startingmaterial from which the cells originated; however, it differssignificantly from that of adult and osteoarthritic articular cartilagein that collagen types I, III and X were not detected.

Collagens of the neocartilage matrix can be isolated via pepsinizationand neutral salt precipitation (Miller, E J and Rhodes R K; 1982,Methods of Enzymology 82:33-64) for use in the production of cartilagebiomatrices. The culture conditions described herein are ideal forproducing the normal complement and ratio of cartilage collagens, whichinclude but are not limited to types II, VI, IX, and XI.

EXAMPLE 3 Model Systems for Studying Articular Cartilage Disease andArticular Cartilage Response to Natural and Synthetic Compounds

Neocartilage cultures were established in 24 or 48 well plastic dishes(Corning) as described above (Example 1) and grown to day 30. Thehyaline cartilage matrix was then stimulated to undergo autolyticdegradation following 30 day activation with increasing concentrationsof a variety of cytokine and/or inflammatory agents, including IL-1,Il-6, IL-17, TNF-α, LPS, and phorbol ester. Culture media were collectedevery 48 hours and subsequently changed by the addition of freshmedia±inflammatory stimuli.

Cultures were terminated and the biochemical composition of theneocartilage matrix that remained was analyzed as described previously.Parallel samples were processed for light and transmission electronmicroscopy for histological evaluation of extracellular matrixcomponents.

Conditioned media were thawed and identification of chondrocyte-derivedmetalloproteinases assessed by zymogen substrate gel electrophoresis, aswell as Western analysis following SDS-PAGE. Further studies examinedthe effect of activating agents on synthesis of S-GAG and collagen viaradiolabeled incorporation.

Resorption of neocartilage material was readily apparent by grossexamination between day 20-25 of stimulation (FIG. 8). Resorption wascharacterized by a reduction in the diameter of the neocartilagematerial and its retraction upon removal from the culture vessel,indicating that the structural integrity of the tissue was significantlycompromised.

Histological evaluation (i.e., safranin-O and pentachrome staining) oftreated and control material revealed a marked reduction in the S-GAGand collagen content of treated samples (FIG. 9). TEM studies alsodemonstrated a significant alteration in the morphological appearance ofthe chondrocytes to more of a fibroblast/macrophage lineage (i.e.,numerous microvillus projections were identified on the surface offlattened, spindle shaped cells), whereas chondrocytes in untreatedcontrols maintained their rounded phenotype (not shown).

The OH-proline and S-GAG content of treated neocartilage disks werereduced by 20-85% depending on the dose and identity of the activatingagent (Table II). Both zymogram and Western analysis identifiedcollagenolytic and casienase activities that were upregulated in thepresence of activating agent alone (FIG. 10).

TABLE II Hydroxyproline content of tissue matrix following chroniccytokine treatment OH-proline content Condition of tissue (pmoles)*Control 1710 ± 251 +50 mU plasminogen‡ 1648 ± 72  IL-1β 0.1  572 ± 105(ng/ml) 1.0 541 ± 16 5.0 501 ± 48 +50 mU plasminogen 460 ± 16 TNF-α 1.0403 5.0 355 *Samples were lyophilized and digested with papain prior tohydrolysis for determination of OH-proline content via HPLC analysis.‡Human plasminogen was added to enhance the activation of latent MMPsvia the plasminogen activator/plasmin cascade.

Phorbol ester specifically induced synthesis of gelatinase B, whereasthe cytokine treatments resulted in the production of numerous bands(9-11) on substrate gels, three of which were identified as collagenase1, 2 and 3 by Western immonoblotting (FIG. 11). Each of these enzymesare implicated in arthritic disease.

This system is unique in that the anabolic and catabolic properties ofnormal human chondrocyte metabolism can be examined, under definedserum-free conditions. It is clear that the described model ofneocartilage formation will facilitate the development and evaluation ofpharmacological agents to protect the cartilage matrix from arthriticdestruction.

EXAMPLE 4 Surgical Repair of Rabbit Articular Cartilage viaTransplantation of Rabbit Neocartilage Allografts

neocartilage allografts were produced from 10-day neonates as describedin Example 1. Extreme care was taken to eliminate the use ofvascularized epiphyseal cartilage during tissue preparation.

Neocartilage implants were grown to day 30, fixed and stained forhistological evaluation, and further extracted for analysis of cartilagespecific macromolecules. This procedure yielded hyaline tissue that wasenriched in both cartilage specific collagens and glycosaminoglycans(Table III).

TABLE III Composition of Rabbit Neocartilage Relative ComponentAbundance Collagen type I − type II +++ type IX + type XI + Aggrecan14,365 ± 410 μg High density cultures were established in 12 wellclusters as described, and neocartilage formation was allowed to proceeduntil harvest on day 40. Triplicate samples were assayed for total S-GAGusing dimethyl methylene blue. Isotyping of collagen was performed viaelectrophoresis on 6.5% SDS-polyacrylamide gels under reducedconditions.

Eighteen (18) skeletally mature (30 wk) male New Zealand White rabbitswere divided into three groups to assess the healing potential oftransplanted neocartilage at 1, 3, 6 and 12 weeks post-operatively.Rectangular defects of approximately 3 mm in width, spanning the girthof the medial femoral condyle (5 mm), were created surgically in bothknees (FIG. 12). Violation of subchondral bone was avoided during thisprocedure.

Sterile neocartilage implants (day 35-45 in vitro) were subsequently cutto size and sutured into the experimental defect (right side) using 7-0vicryl suture, anchoring neocartilage to the medial and lateralperichondria, following addition of tissue transglutaminase as describedin Jurgensen et al., J. Bone Joint Surg. 79-A, 185-193 (1997). Unfilleddefects (left side) were allowed to heal intrinsically and served as thecontralateral sham control. Arthrotomies were repaired using 4-0 vicrylsuture and animals were permitted free cage activity. Oral analgesic wasprovided for 24 hours post-operatively as needed for pain.

The animals showed excellent tolerance of the surgical procedures,displaying normal ambulation and increased appetite within 24 hourspost-operatively. Gross examination of the experimental defectsharvested at six weeks revealed good adherence of grafts to surroundingtissue, whereas unfilled defects remained unfilled (FIG. 13).

EXAMPLE 5 Immunological Assessment of Allograft Rejection

A one-way mixed leukocyte reaction (MLR) was set up in whichchondrocytes, acting as the stimulator population, were firstgamma-irradiated in order to block their proliferative potential.Allogeneic leukocytes, obtained from either peripheral blood or buffycoats (American Red Cross) were then passed over a Ficoll gradient. Thispopulation of mononuclear leukocytes was counted and subsequentlyco-cultured with chondrocytes, having first been isolated from day 30-45neocartilage allografts. Adult cells were also isolated from day 30cultures which were grown under conditions identical to theneocartilage.

Proliferation of non-irradiated leukocytes was determined on day 7,following a 24 hr pulse with tritiated thymidine (Amersham, 1 μCi/ml)(Abbas, A K, et al. 1991 In Cellular and Molecular Immunology, pp.320-322). Culture plates were frozen, the cells lysed in water, and thenuclear DNA aspirated and bound to glass filtermats using an automatedcell harvester. Filtermats were then dried and counted in a WallacMicroBeta scintillation counter. Chondrocytes isolated from day 30-45neocartilage failed to generate an MLR response that would indicate across match (FIG. 14B), while the positive controls showed a 19-foldincrease in proliferation.

These data support the concept that chondrocytes are immunologicallyprivileged. Although chondrocytes are reported to present MHC class IIantigens on their cell surface [Elves, J. Bone Joint Surg., 56B:178-185(1974); Jahn et al., Arth Rheum 30:64-74 (1987); and Jobanputra et al.,Clin. Exp. Immunol. 90:336-344 (1992)], proliferation of the responderleukocyte population was not detected in three separate assays involvingat least twelve (12) different neocartilage samples. From animmunological perspective, these experiments indicate that the presentapproach to cartilage repair (e.g., allograft transplantation) isfeasible.

Additional studies examining the co-stimulatory function of chondrocytesin a T-cell based assay (Abbas, A K, et al. 1991, In Cellular andMolecular Immunology, pp. 320-322) were also negative, indicating thatco-stimulatory molecules, probably B7.1 (CD80) and B7.2 (CD86) are notnormally expressed by chondrocytes (FIG. 15). Thus, applicant hasdiscovered appropriate culture conditions which permit transplantationof neocartilage into allogeneic recipients.

EXAMPLE 6 Overlaying of Chondrocytes to Increase the Thickness andStructural Integrity of Neocartilage

Rabbit and human chondrocytes were isolated, seeded into 12-well dishesand grown to day 10 as described above. During the initial plating ofthese cultures, 1.4×10⁷ cells were reserved for plating in 100 mmdishes. Cells were subsequently released from the culture surface on day10 using the collagenase/hyaluronidase procedure described in Example 1.

Chondrocytes were filtered, counted and resuspended in a 1:1 mixture ofDMEM+10% FBS/Hl-1, containing ascorbate, at a density of 0.5×10⁶/ml. Day10 cultures (12-well plates) were overlayed with 1 ml of the cellsuspension to increase the thickness and structural integrity of theneocartilage grafts. The cultures were maintained in the 1:1 mix ofDMEM/HL-1 from day 13-17, after which they were grown in HL-1 mediauntil harvest at day 40. Total S-GAG in the neocartilage matrix werequantified as described above. FIG. 16 shows that the overlayingprocedure increased total S-GAG content by approximately 50%, and as aresult increased the rigidity of the neocartilage matrix.

EXAMPLE 7 Isolation and Characterization of Neocartilage Proteoglycan

Neocartilage cultures were established and grown to day 35 as describedin Example 1. Newly synthesized proteoglycans were metabolically labeledfor 72 hrs using carrier-free [³⁵S]-sodium sulfate (55 μCi/ml, Amersham,supra). Following three washes with PBS to remove unincorporated label,the proteoglycans of the neocartilage matrix were extracted in 4 Mguanidine and precipitated in ethanol. Unlabeled native proteoglycanswere also extracted from the articular cartilage of a 12-yr female and43-yr male for comparison. Twenty (20) μg of S-GAG were loaded andseparated on 1.2% agarose gels according to the method of Bjornsson,Anal. Biochem., 210: 292-298 (1993), (FIG. 17).

Greater than 95% of the labeled material was identified as aggrecan, asjudged by molecular weight. However, it was noted that this material wasof higher molecular weight than the aggrecan found in pre-teen and adulttissue, indicating that the aggrecan monomers were more highlyglycosylated. A minor band with electrophoretic mobility identical tothat of native decorin was also observed in the neocartilage matrix.Biglycan, present as a minor component of native articular cartilage,was not identified via radiolabel incorporation in 6 replicates ofneocartilage. Only 12-15% of the total radiolabeled S-GAG could berecovered in culture supernatant, generating the same profile as thatshown here.

Thus, the composition of neocartilage proteoglycan differs from that ofmature articular cartilage in two distinct ways. First, biglycan was notidentified as a component of the neocartilage matrix; and second, theinnate composition of neocartilage proteoglycan appears to be morehighly glycosylated, resulting in a high molecular weight aggrecan. Itis believed that this aggrecan may play a role in tissue growth andrepair by binding specific growth factors with greater avidity thanadult aggrecan.

Purification of this material for testing as a wound healing agent isrelatively easy and inexpensive. High molecular weight aggrecan bandsidentified on 1.2% agarose gels were removed and eluted to purity usinga BioRad electroelution device. Preliminary studies demonstrate that a200 mg neocartilage disk yielded 3-4 mg of high molecular weightaggrecan.

EXAMPLE 8 Role of Heparin Binding Proteins in Neocartilage Formation:Identification of Two New Members of the TGF-β Superfamily

Fetal chondrocytes were grown as described above and subdivided intothree groups on day 10 to investigate the effect of heparin onneocartilage formation. In low doses, heparin is known to increase theactivity of certain growth factors, for example the fibroblast growthfactor (FGF) family of related proteins, by acting as a co-factor,whereas greater amounts of heparin will strip away certain growthfactors from the extracellular matrix and thereby impede tissue repair.

Sterile heparin was added to the media at a final concentration of 0.2,2.0, and 20 μg for the duration of the culture period (i.e. from day10-28). Media were exchanged every 3-4 days and recovered forelectrophoretic analysis of secreted proteins. Cell layers were removedand the S-GAG (FIG. 18A), hydroxy proline (FIG. 18B), and DNA contentquantitated as before (FIG. 18). The highest concentration of heparincaused a severe defect (60% inhibition in S-GAG and 33% in OH-proline)in matrix deposition. This effect was also evident in the 2 μg/mltreatment group, but was much less severe.

When these heparin binding proteins are then extracted from theneocartilage matrix using procedures adapted from Chang et al.,approximately 10 proteins showing a molecular mass of less than 60 kDaare visible on a 4-20% gradient Tris/Tricine polyacrylamide gel (FIG.19). Western blotting using antibodies from the NIH have shown thatcartilage derived morphogenetic protein-1 and -2 (CDMP-1 and -2) arepresent, as was connective tissue derived growth Factor (CTGF) (notshown).

EXAMPLE 9 Serum-Free Culture Depletes Neocartilage of Precursor FattyAcids for Eicosanoid Synthesis

Adkisson et al., FASEB J 5: 344-353 (1991), discovered highconcentrations of 20:3 n-9 eicosatrienoic acid and low concentrations ofn-6 polyunsaturated fatty acids (PUFA) in normal, rapidly growingcartilages of several vertebrates, a biochemical hallmark of essentialfatty acid deficiency. Because the n-6 fatty acids, in particulararachidonic acid (20:4 n-6), are key mediators of inflammation andtransplant rejection (Schreiner et al., Science 240: 1032-1033 (1988),the fatty acid composition of neocartilage grafts were characterizedduring a 28-day time course in vitro.

Total cellular lipids and the phospholipid fraction were isolated asdescribed in Adkisson et al., supra. Pentafluorobenzyl ester derivativesof the free fatty acids obtained from the phospholipid fraction wereprepared for microanalysis by capillary gas chromatography using aSP-2380 fused silica column (Supelco Inc., Bellefonte, Pa.). The gaschromatograph (HP model 5890) was held at 160° C. for 2 min.,temperature programmed at 10° C./min to 200° C., and maintained at thistemperature for an additional 10 min. Both the injector and theelectrochemical detector temperatures were set at 225° C. Derivatizedfatty acids were identified by co-migration with authentic standards,while unusual fatty acids were characterized by their fragmentationpattern using mass spectrometry analysis of their picolinyl esterderivative as described previously (Adkisson et al., 1991). Percentcomposition of individual fatty acids was determined by integrationusing Hewlett-Packard model 3393A integrator.

FIG. 20 illustrates that essential fatty acids, a component of theculture media (i.e., 5-10% FBS from day 1-10), are rapidly incorporatedinto the phospholipid stores of isolated chondrocytes. Moreover, it isclear that once the cultures are switched to and maintained inserum-free HL-1 media (closed circles), the native complement of fattyacids is restored, such that low levels of the n-6 PUFAs are detected.The n-6 fatty acids, shown in the upper three panels, continue toaccumulate in chondrocyte phospholipids when the neocartilage culturesare maintained in serum-containing media (open circles).

FIG. 21 is a mass chromatogram of the dominant polyunsaturated fattyacid identified in neocartilage phospholipids (i.e. 20:3 n-9eicosatrienoic acid) at day 28 of culture. The fragmentation patternmatches that of authentic 20:3 n-9 eicosatrienoic or Mead Acid.

It is unclear whether enrichment in n-9 fatty acids reduces antigenexpression of MHC class II molecules or B7 co-stimulatory moleculeswhich are required to establish immunogeneity in allograft rejection.The literature does support the fact that a dearth of n-6 PUFA modulatesthe infiltration of inflammatory cells by inhibiting leukotrieneformation (Cleland et al., 1984, Lipids 29:151-155, and Schreiner etal., 1988, supra). This in turn protects organs from possible transplantrejection.

In summary, the novel serum-free culture system used to produceneocartilage tissue may facilitate allogeneic transplantation of theneocartilage by regulating chondrocyte immunogenicity, as well as theelaboration of inflammatory eicosanoids/cytokines.

EXAMPLE 10 Preparation of Neocartilage/Demineralized Bone Composites

Day 60 neocartilage cultures were enzymatically dispersed as describedin Example 1. One million chondrocytes were then seeded onto samples ofdemineralized allograft bone (Lambone™, 100-300 μm thick) which werefirst trimmed to accomodate in vitro culture in 12 well clusters.Lambone™, obtained from Pacific Coast Tissue Bank, Los Angeles, Calif.,was washed extensively in culture media to remove residual ethyleneoxide gas used during tissue processing.

Neocartilage formation on demineralized allograft bone was allowed toproceed to day 28 as described herein. Neocartilage/Lambone compositeswere then rinsed and fixed overnight in 10% neutral buffered formalinfor morphological examination via pentachrome staining.

FIGS. 22A and B illustrate the morphologic appearance ofneocartilage/Lambone™ composites following pentachrome staining.Magnification: 22A, 100x; 22B 200x.

In view of the above, it will be seen that the several objects of theinvention are achieved.

As various changes could be made in the above compositions and methodsby the person skilled in the art after reading the present disclosurewithout departing from the spirit and scope of the invention, it isintended that all matter contained in the above description shall beinterpreted as illustrative of the claimed invention and not in alimiting sense.

What is claimed is:
 1. A pharmaceutical composition comprising aneffective amount of cartilage substantially free of biglycan havingmultiple layers of cells randomly organized, rather than separated intodistinct zones of chondrocyte maturation, surrounded by a substantiallycontinuous insoluble glycosaminoglycan and collagen enriched hyalineextracellular matrix.
 2. A pharmaceutical composition as set forth inclaim 1 wherein the membrane phospholipids are enriched in Mead acid. 3.A pharmaceutical composition as set forth in claim 2 wherein the Meadacid content of said membrane phospholipids comprises at least about0.4% of the total fatty acid content of said membrane phospholipids. 4.A pharmaceutical composition comprising an effective amount of cartilagecontaining membrane phospholipids depleted in linoleic acid.
 5. Apharmaceutical composition as set forth in claim 4 wherein the linoleicacid content of said membrane phospholipids comprises less than about0.5% of the total fatty acid content of said membrane phospholipids. 6.A pharmaceutical composition as set forth in claim 4 wherein thelinoleic acid content of said membrane phospholipids comprises less thanabout 0.2% of the total fatty acid content of said membranephospholipids.
 7. A pharmaceutical composition comprising an effectiveamount of cartilage containing membrane phospholipids depleted inarachidonic acid.
 8. A pharmaceutical composition as set forth in claim7 wherein the arachidonic acid content of said membrane phospholipidscomprises less than about 0.5% of the total fatty acid content of saidmembrane phospholipids.
 9. A pharmaceutical composition as set forth inclaim 7 wherein the arachidonic acid content of said membranephospholipids comprises less than about 0.2% of the total fatty acidcontent of said membrane phospholipids.
 10. A pharmaceutical compositionas set forth in claim 2 wherein said cartilage is substantially free ofendothelial, bone and synovial cells.
 11. A pharmaceutical compositionas set forth in claim 2 wherein said cartilage has a S-GAG content of atleast about 400 mg/mg of OH-proline.
 12. A pharmaceutical composition asset forth in claim 2 wherein said cartilage has a S-GAG content of fromabout 800 mg to about 2500 mg/mg of OH-proline.
 13. A pharmaceuticalcomposition as set forth in claim 2 wherein said cartilage issubstantially free of type I, III and X collagen.
 14. A pharmaceuticalcomposition comprising an effective amount of cartilage containing amatrix substantially free of biglycan.
 15. A pharmaceutical compositionas set forth in claim 2 comprising cartilage enriched in high molecularweight aggrecan.
 16. A pharmaceutical composition as set forth in claim15 wherein the high molecular weight aggrecan comprises at least about80% of the total proteoglycan content of said cartilage.
 17. Apharmaceutical composition as set forth in claim 15 wherein the highmolecular weight aggrecan comprises about 90% of the total proteoglycancontent of said cartilage.
 18. A pharmaceutical composition comprisingan effective amount of cartilage characterized as follows: (a)containing membrane phopholipids enriched in Mead acid and depleted inlinoleic and arachidonic acid; (b) being enriched in high molecularweight aggrecan; (c) being substantially free of endothelial, bone andsynovial cells; (d) being substantially free of biglycan.
 19. Apharmaceutical composition as set forth in claim 18 wherein saidcartilage is further characterized by having multiple layers of cellssurrounded by a continuous insoluble glycosaminoglycan and collagenenriched hyaline extra cellular matrix containing no biglycan.
 20. Apharmaceutical composition as set forth in claim 1 wherein saidcartilage is further characterized by having multiple layers of cellssurrounded by a substantially continuous insoluble glycosaminoglycan andcollagen enriched in hyaline extracellular matrix depleted in bothlinoleic acid and arachidonic acid.
 21. A pharmaceutical composition asset forth in claim 1 wherein said cartilage is substantially free oftypes I, III, and X collagen.
 22. A pharmaceutical composition as setforth in claim 1 wherein said cartilage is further characterized byhaving membrane phospholipids which are depleted in linoleic acid.
 23. Apharmaceutical composition as set forth in claim 1 wherein saidcartilage is further characterized by having membrane phospholipidswhich are arachidonic acid.
 24. A pharmaceutical composition as setforth in claim 1 wherein said cartilage is substantially free ofendothelial, bone and synovial cells.
 25. A pharmaceutical compositionas set forth in claim 1 wherein said cartilage is enriched in highmolecular weight aggrecan.
 26. A pharmaceutical composition as set forthin claim 1 wherein said cartilage is substantially free of type Xcollagen.
 27. A pharmaceutical composition comprising an effectiveamount of cartilage containing membrane phospholipids and havingmultiple layers of cells randomly organized, rather than separated intodistinct zones of chondrocyte maturation, wherein: (a) said membranephospholipids are enriched in Mead acid and depleted in linoleic andarachidonic acid; (b) said cartilage is enriched in high molecularweight aggrecan; (c) said cartilage is substantially free ofendothelial, bone, and synovial cells; and (d) said cartilage issubstantially free of biglycan.
 28. A pharmaceutical composition as setforth in claim 27 wherein said cartilage is substantially free of typeIII and type X collagen.
 29. A pharmaceutical composition as set forthin claim 27 wherein said cells are further characterized as beingsurrounded by a substantially continuous insoluble glycosaminoglycan andcollagen enriched hyaline extracellular matrix.
 30. A pharmaceuticalcomposition as set forth in claim 27 wherein said cartilage issubstantially free of type I, III, and X collagen.
 31. A pharmaceuticalcomposition comprising an effective amount of cartilage enriched in Meadacid and substantially free of endothelial, bone, and synovial cells.32. A pharmaceutical composition comprising an effective amount ofcartilage enriched in Mead acid and depleted in linoleic and arachadonicacid.
 33. A pharmaceutical composition comprising an effective amount ofcartilage enriched in high molecular weight aggrecan and in Mead acid.34. A pharmaceutical composition comprising an effective amount ofcartilage substantially free of endothelial, bone and synovial cells andtype I, III, and X collagen.
 35. A pharmaceutical composition comprisingan effective amount of cartilage enriched in high molecular weightaggrecan and substantially free of endothelial, bone, and synovialcells.
 36. A pharmaceutical composition as set forth in claim 27 whereinthe high molecular weight aggrecan comprises about 80% of the totalproteoglycan content of said cartilage.
 37. A pharmaceutical compositionas set forth in claim 27 wherein the high molecular weight aggrecancomprises about 90% of the total proteoglycan content of said cartilage.38. A pharmaceutical composition as set forth in claim 27 wherein saidcartilage has a S-GAG content of at least about 800 mg/mg of OH-proline.39. A pharmaceutical composition as set forth in claim 27 wherein saidcartilage has a S-GAG content of from about 800 mg to about 2500 mg/mgof OH-proline.